1. Field of the Invention
This invention relates generally to a recombinant adenoviral vector construct and to methods for the study of gene function and gene therapy for heart disease and more specifically to methods of targeting tissue specific expression of a given transgene in cardiac tissue through use of inverted terminal repeat sequences from human adeno-associated virus.
2. Background Information
Cardiovascular gene therapy represents a novel approach to the treatment of inherited and acquired heart disease. Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that are responsible for proper cardiac function. The control of in vivo cardiac function represents a complicated interplay between multiple genes, varied cell types, and environmental stimuli but the elucidation of this interplay remains dependent on a more complete understanding of the changes that occur at the molecular and cellular levels. Traditionally, the majority of human gene therapy protocols have relied on the ex vivo application of the therapeutic gene, through the introduction of a retroviral vector, to the affected cells or tissue. Because the ex vivo method of gene therapy depends on the removal from and reintroduction to the body of the target cells, the treatment of inaccessible or sensitive organs or tissue poses a major dilemma. The alternate strategy of direct in vivo delivery of therapeutic genes to the target cells represents a preferable method of gene therapy.
Targeted gene expression in somatic tissues is essential for both gene therapy and in vivo analysis of gene function, mainly through the substitution of an affected gene, using a safe and effective delivery system for the therapeutic gene. To date, recombinant adenoviruses have replaced the retrovirus as an efficient gene delivery vector for a variety of cell types and tissues (Yeh, et al., FASEB J 11, 615-23, 1997). Adenovirus vectors are highly efficient in the genetic modification of nondividing human cells and have the capacity to carry long segments of genetic information. The hurdle in using adenovirus as gene xe2x80x9cdelivery systemsxe2x80x9d is that when an adenovirus is administered to a patient to aid in the delivery of genes to specific cells, the patient""s immune system may react against the virus. To overcome this hurdle, modifications have been made to make the adenoviral vector safer, less toxic to the cells and less likely to stimulate an immune response. This has involved removing the E1 region of the adenovirus gene which prevents the ability of the virus to express its own proteins required for making viral particles. In place of the E1 region, a therapeutic transgene can be inserted. The efficiency of this kind of exogenous gene delivery and subsequent expression can be high, as it does not normally integrate into the host genome, and it has a minimal effect on intrinsic host cell function (Baldwin, et al., Gene Ther. 4, 1142-49, 1997). However, while adenoviral vectors are capable of producing high levels of transgene expression, their capacity to infect and program transgene expression in large numbers of cells and tissue, including the liver and lungs, poses limitations. As a result of this high level of transient infectivity, methods have been undertaken to direct transgene expression to specific tissues or areas of the body. For cardiac tissue, a number of attempts have been reported utilizing recombinant adenoviruses to achieve transgene expression in the heart through either intra-myocardial or intra-coronary injection (Brody, et al., Ann. N.Y. Acad. Sci. 716, 90-101, 1994; Barr, et al., Gene Ther. 1, 51-8, 1994; Kypson, et al., J. Thorac. Cardiovasc. Surg. 115, 623-30, 1998). While direct injection of viral particles into the myocardium or cardiac cavity have been shown to be more efficient for gene delivery to the myocardium, infection and transgene expression also occurs in non-cardiomyocytes, which causes speculation that any specificity of transgene expression that exists is achieved by targeted delivery rather than restricted transcription (Kass, et al., Gene Ther. 1, 395-402, 1994; Kass, et al., Methods Cell Bio. 52, 423-37, 1997). As a result, ectopic expression, particularly in liver and other tissue, remains a significant limitation for the generalized use of recombinant adenoviruses for gene transfer to specific cell types within the cardiovascular and other organ systems.
In most recombinant adenoviral vectors, the E1a region of the adenovirus genome, which encodes the protein with properties for transcriptional regulation, is deleted and replaced by a minigene xe2x80x9ccassettexe2x80x9d that typically includes a promoter of choice, the transgene coding region, and a polyadenylation signal (Yeh, et al., FASEB J 11, 615-23, 1997). One possible approach to achieve tissue-specific transgene expression using adenoviruses is to employ cellular gene promoters that possess cell-type specificity at the transcriptional level, rather than commonly used viral gene promoters that have a high level of expression, but lack tissue specificity. In the past, a number of studies have utilized different cell promoters to achieve targeted transgene expression in various tissues, including smooth muscle (Kim, et al., J. Clin. Invest. 100, 1006-14, 1997), pancreas (Dusetti, et al., J. Biol. Chem. 272, 5800-4, 1997), endothelium (Morishita, et al., J. Biol. Chem. 270, 27948-53, 1995), lung (Strayer, et al., Am. J. Respir. Cell Mol. Bio. 18, 1-11, 1998), and several kinds of tumors ( Su, et al., Proc. Natl. Acad. Sci. USA 94, 13891-6, 1997; Siders, et al., Cancer Res. 56, 5638-46, 1996). Similar attempts using cardiac-specific promoters such as the myosin light chain-2 (MLC-2v) and the alpha-myosin heavy chain (xcex1-MHA) promoters, in the context of adenoviruses, however, have not been wholly successful in providing tissue-restricted gene expression in vivo (Kim, et al., J. Clin. Invest. 100, 1006-14, 1997). These results suggest that adenoviral genomic sequences surrounding the deleted E1a region may be responsible for at least partial specificity of the adjacent cellular promoter. It has also been suggested that sequences around the E1a region may contain negative regulatory elements that act in modulating the specificity and activity of a cellular promoter (Shi, et al., Hum. Ther. 8, 403-10, 1997). This undesirable property of adenoviral vectors has limited their application, especially in the context of in vivo studies where tissue specific expression of the transgene is required.
Thus, the need remains for a transgene expression system utilizing recombinant adenoviral vectors that are tissue specific for use in in vivo and in vitro gene therapy and gene function analysis for both neonatal and adult subjects. The present invention satisfies this need and provides related advantages as well.
The present invention provides a human type-5 recombinant adenovirus vector to achieve cardiac restricted transcription in both neonatal and adult subjects utilizing the cardiomyocyte-restricted cardiac ankyrin repeat protein (CARP) promoter in cooperation with the inverted terminal repeat (ITR) sequences from human adeno-associated virus (AAV). Such a combination is effective in achieving cardiac tissue-specific transcription of the transgene both in vitro and in vivo.
The invention further provides a method to achieve tissue targeted expression of a given transgene in cardiac tissues in both neonatal and adult subjects. Such a method has significant applications in both gene function studies and gene therapy for inherited and acquired heart diseases.